An investigation into the multiple coupling capacity of prostacyclin receptors.

Kam Yiu-wing.Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.Includes bibliographical references (leaves 200-215).Abstracts in English and Chinese.Abstract --- p.iAcknowledgements --- p.iiiPublications --- p.ivAbbreviations --- p.vContents --- p.viiChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- G protein-coupled receptors --- p.1Chapter 1.1.1 --- Introduction --- p.1Chapter 1.1.2 --- G protein-coupled receptors --- p.2Chapter 1.1.3 --- Heterotrimeric G-proteins --- p.4Chapter 1.1.4 --- Second messenger systems --- p.5Chapter 1.1.5 --- Mechanism of GPCR activation --- p.6Chapter 1.1.6 --- GPCR cross talk --- p.8Chapter 1.2 --- Receptor theory --- p.10Chapter 1.2.1 --- Introduction --- p.10Chapter 1.2.2 --- Two-state model --- p.11Chapter 1.2.3 --- Three-state model --- p.12Chapter 1.2.4 --- Extended ternary complex model --- p.12Chapter 1.2.5 --- Multiple receptor state --- p.13Chapter 1.2.6 --- Constitutively active mutant receptors --- p.14Chapter 1.3 --- Agonist trafficking --- p.15Chapter 1.3.1 --- Introduction --- p.15Chapter 1.3.2 --- Effect of agonist efficacy on receptor coupling --- p.17Chapter 1.3.3 --- Effect of receptor expression level on receptor coupling --- p.17Chapter 1.3.4 --- Receptor promiscuity --- p.18Chapter 1.3.5 --- Agonist-induced conformational changes --- p.19Chapter 1.3.5.1 --- Conformational induction --- p.19Chapter 1.3.5.2 --- Conformational selection --- p.19Chapter 1.3.6 --- Receptor/G-protein system --- p.20Chapter 1.3.7 --- Implication of agonist trafficking --- p.21Chapter 1.4 --- Receptor switching --- p.22Chapter 1.4.1 --- Introduction --- p.22Chapter 1.4.2 --- Receptor switching --- p.23Chapter Chapter 2 --- Prostacyclin receptors --- p.34Chapter 2.1 --- General properties of prostacyclin --- p.34Chapter 2.1.1 --- Synthesis of prostacyclin --- p.34Chapter 2.1.2 --- Prostacyclin and its mimetics --- p.34Chapter 2.1.3 --- Characterization of IP-receptors --- p.36Chapter 2.1.3.1 --- Classification of IP-receptors --- p.36Chapter 2.1.3.2 --- Distribution of IP-receptors in the peripheral system --- p.36Chapter 2.1.3.3 --- Distribution of IP-receptors in the central nervous system --- p.38Chapter 2.1.3.4 --- Structure of IP-receptors --- p.39Chapter 2.1.4 --- Anti-thrombotic role of prostacyclin --- p.40Chapter 2.1.5 --- Cytoprotective role of prostacyclin --- p.41Chapter 2.1.6 --- Role of prostacyclin in adipogenesis --- p.42Chapter 2.2 --- Coupling capacity of IP-receptors --- p.43Chapter 2.2.1 --- Introduction --- p.43Chapter 2.2.2 --- Interaction with Gs --- p.44Chapter 2.2.3 --- Interaction with Gi --- p.45Chapter 2.2.4 --- Interaction with Gq --- p.45Chapter 2.2.5 --- Interaction with PPARs --- p.43Chapter 2.2.6 --- IP-receptor isoprenylation --- p.49Chapter 2.3 --- Regulation of IP-receptors --- p.50Chapter 2.3.1 --- Mechanism of signal termination --- p.50Chapter 2.3.2 --- Desensitization of IP-receptors --- p.51Chapter 2.3.3 --- Internalization of IP-receptors --- p.53Chapter Chapter 3 --- Materials and solutions --- p.59Chapter 3.1 --- Materials --- p.59Chapter 3.2 --- "Culture media, buffers and solutions" --- p.53Chapter 3.2.1 --- Culture media --- p.63Chapter 3.2.2 --- Buffers --- p.64Chapter 3.2.3 --- Solutions --- p.65Chapter Chapter 4 --- Methods --- p.67Chapter 4.1 --- Cell culture --- p.67Chapter 4.2 --- Mammalian cell transfection --- p.68Chapter 4.2.1 --- Preparation of plasmid DNA --- p.68Chapter 4.2.2 --- Transient transfection of mammalian cells --- p.71Chapter 4.2.3 --- β-galactosidase assay --- p.73Chapter 4.2.3.1 --- Introduction --- p.73Chapter 4.2.3.2 --- Preparation of cell lysate --- p.73Chapter 4.2.3.3 --- Micro β-galactosidase assay --- p.73Chapter 4.3 --- Measurement of adenylate cyclase activity --- p.74Chapter 4.3.1 --- [3H]-adenine prelabelling method --- p.74Chapter 4.3.1.1 --- Preparation of columns --- p.74Chapter 4.3.1.2 --- Incubation of cells --- p.75Chapter 4.3.1.3 --- Measurement of [3H]-cyclic AMP production --- p.75Chapter 4.3.1.4 --- Data analysis --- p.76Chapter 4.3.2 --- cAMP [125I]-scintillation proximity assay (SPA) --- p.77Chapter 4.3.2.1 --- Introduction --- p.77Chapter 4.3.2.2 --- Cell lysis method --- p.77Chapter 4.3.2.3 --- cAMP [I25I]-scintillation proximity assay --- p.78Chapter 4.4 --- Measurement of phospholipase C activity --- p.78Chapter 4.4.1 --- Introduction --- p.78Chapter 4.4.1.1 --- Preparation of columns --- p.78Chapter 4.4.1.2 --- [3H]-inositol phosphate assay --- p.79Chapter 4.4.1.3 --- Measurement of [3H]-inositol phosphate production --- p.79Chapter 4.4.1.4 --- Data analysis --- p.80Chapter 4.4.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.81Chapter 4.4.2.1 --- Introduction --- p.81Chapter 4.4.2.2 --- Sample preparation --- p.81Chapter 4.4.2.3 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.82Chapter 4.5 --- Measurement of changes in intracellular Ca2+ concentration --- p.82Chapter 4.5.1 --- Introduction --- p.82Chapter 4.5.2 --- Cell preparation --- p.83Chapter 4.5.3 --- Measurement of Fura-2 fluorescence --- p.83Chapter 4.6 --- Radioligand binding --- p.84Chapter 4.6.1 --- Introduction --- p.84Chapter 4.6.2 --- [3H]-iloprost ligand binding --- p.85Chapter 4.6.3 --- Data analysis --- p.86Chapter 4.7 --- Cytotoxicity test using trypan blue exclusion test --- p.86Chapter Chapter 5 --- Multiple coupling capacity of prostacyclin receptors in CHO cells --- p.88Chapter 5.1 --- Introduction --- p.88Chapter 5.2 --- Method --- p.88Chapter 5.3 --- Results and Discussion --- p.89Chapter 5.3.1 --- IP agonist log concentration-response curves for [3H]-cAMP and [3H]-inositol phosphate production in mIP-CHO cells --- p.89Chapter 5.3.2 --- Effect of varying Gαs cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.90Chapter 5.3.3 --- Effect of varying Gαq cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.92Chapter 5.4 --- Conclusion --- p.95Chapter Chapter 6 --- Multiple coupling capacity of prostacyclin receptors in neuroblastoma cells --- p.113Chapter 6.1 --- Introduction --- p.113Chapter 6.2 --- Method --- p.114Chapter 6.3 --- Results and Discussion --- p.114Chapter 6.3.1 --- Characterization of prostanoid receptors in SK-N-SH cells --- p.114Chapter 6.3.2 --- Property of IP-receptor signaling in SK-N-SH cells --- p.116Chapter 6.3.3 --- Effect of Gαq subunits on PLC activation in SK-N-SH cells --- p.117Chapter 6.3.4 --- Coupling capacity of IP-receptorin rat/mouse neuroblastoma (NG108-15) cells --- p.119Chapter 6.4 --- Conclusion --- p.123Chapter Chapter 7 --- Agonist trafficking --- p.133Chapter 7.1 --- Introduction --- p.133Chapter 7.2 --- Method --- p.134Chapter 7.3 --- Results and Discussion --- p.134Chapter 7.3.1 --- Simultaneous measurement of AC and PLC activation in metabolically-labelled mIP-CHO cells --- p.134Chapter 7.3.1.1 --- Effect of IBMX on PLC activation --- p.135Chapter 7.3.1.2 --- Effect of Li+ ion on AC activation --- p.135Chapter 7.3.1.3 --- Separation of [3H]-adenine and [3H]-inositol using column chromatography method --- p.136Chapter 7.3.2 --- Measurement of AC and PLC activation using different assay systems --- p.137Chapter 7.3.2.1 --- cAMP 125I-Scintillation Proximity Assay System --- p.137Chapter 7.3.2.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H]-assay system" --- p.138Chapter 7.4 --- Conclusion --- p.139Chapter Chapter 8 --- Regulation of prostacyclin receptor coupling --- p.147Chapter 8.1 --- Introduction --- p.147Chapter 8.2 --- Methods --- p.149Chapter 8.3 --- Results and Discussion --- p.150Chapter 8.3.1 --- Role of protein kinases on IP-receptor activity --- p.150Chapter 8.3.2 --- Effect of SQ22536 on IP-receptor activity --- p.155Chapter 8.3.3 --- Role of PKA phosphorylation site in mIP-receptor activity --- p.156Chapter 8.4 --- Conclusion --- p.157Chapter Chapter 9 --- Prostacyclin receptor isoprenylation --- p.171Chapter 9.1 --- Introduction --- p.171Chapter 9.2 --- Method --- p.172Chapter 9.3 --- Results and Discussion --- p.173Chapter 9.4 --- Conclusion --- p.175Chapter Chapter 10 --- IP/DP chimeric receptors --- p.178Chapter 10.1 --- Introduction --- p.178Chapter 10.2 --- Method --- p.179Chapter 10.3 --- Results and Discussion --- p.180Chapter 10.3.1 --- Property of IP/DP chimeric receptors --- p.180Chapter 10.3.2 --- "ID1 chimeric receptor mutant receptors (M4, M5, M6)" --- p.182Chapter 10.3.3 --- "Mutant mIP-receptors (Ml, M2, M3)" --- p.183Chapter 10.3.4 --- Comparison between M5 and ID1 receptors --- p.184Chapter 10.4 --- Conclusion --- p.184Chapter Chapter 11 --- General Discussion and Conclusions --- p.193Chapter 11.1 --- Introduction --- p.193Chapter 11.2 --- Multiple coupling capacity of prostacyclin receptors --- p.193Chapter 11.3 --- Factors influencing prostacyclin receptor coupling --- p.196Chapter 11.4 --- Prostacyclin receptor cross talk and regulation --- p.198References --- p.20